In July 1991, nine bodies were exhumed from a shallow grave just outside Ekaterinburg, Russia. Circumstantial evidence, along with mitochondrial DNA sequencing and matches, gave strong evidence to the remains being those of the Romanovs, the last Russian Royals who were executed on July 18, 1918. This first analysis was made by Peter Gill and his team in UK (Central Research and Support Establishment, Forensic Science Service, Aldermaston, Reading, Berkshire, UK).

Nine skeletons found in a shallow grave in Ekaterinburg, Russia, in July 1991, were tentatively identified by Russian forensic authorities as the remains of the last Tsar, Tsarina, three of their five children, the Royal Physician and three servants. We have performed DNA based sex testing and short tandem repeat (STR) analysis and confirm that a family group was present in the grave. Analysis of mitochondrial (mt) DNA reveals an exact sequence match between the putative Tsarina and the three children with a living maternal relative. Amplified mtDNA extracted from the remains of the putative Tsar has been cloned to demonstrate heteroplasmyat a single base within the mtDNA control region. One of these sequences matches two living maternal relatives of the Tsar. We conclude that the DNA evidence supports the hypothesis that the remains are those of the Romanov family.

*The Tsar's sequence contains a new mutation at position 169, a state referred to as heteroplasmy - the existence of more than one mitochondrial type in the cells of an individual, i.e., the presence of both normal and mutant mtDNA in a single individual. The remains of his brother, Grand Duke of Russia, Georgij Romanov were exhumed, and the results were identical to the Tsar's,including the heteroplasmy at 16169Y. Mitosearch: DQR2R

Tsarina Alexandra, the three children buried with her, and Prince Philip's mitochondrial DNA were an exact match on 740 tested nucleotides. Mitosearch: 2ZACX

Czar Nicholas II, his wife Alexandra and their five children were assassinated as civil war broke out; numerous stories circulated over the years that Anastasia, then 17, somehow escaped. In 1991, five bodies were found and later identified as the Romanovs. Independent studies done in the US and England and later by Rogaev compared extracted DNA samples to those from descendents of the royal family. Two bodies, however, were missing, leaving some question as to the fate of two of the czar's children. Bone fragments were found in the summer of 2007, not far from the original discovery site, about 900 miles east of Moscow, but had been badly damaged by not only time and natural decomposition, but also by acid and fire, as the murderers apparently sought to fully destroy the bodies and evidence of the murder.

In 1890–1891 Nicholas II, then-heir to the throne was on an around-the-world voyage. On 11 May 1891, during his visit to Osaka, Japan, he was attacked and injured in an apparent assassination attempt. The escort policemen swung at Nicholas II’s head with a saber; however the following blow was parried by Prince George of Greece and Denmark who was accompanying Nicholas II. Although the wound was not life-threatening, Nicholas II was severely bleeding and a long scar remained on the right side of his forehead. The Y-chromosome haplotype of the Tsar appears to belong to haplogroup R1b.

Accurate unambiguous identification of ancient or historical specimens can potentially be achieved by DNA analysis. The controversy surrounding the fate of the last Russian Emperor, Nicholas II, and his family has persisted, in part, because the bodies of 2 children, Prince Alexei and 1 of his sisters, have not been found. A grave discovered in 1991 contained remains putatively identified as those of the Russian Royal family. However, not all family members were represented. Here, we report the results of genomic analyses of new specimens, the human remains of 2 burned skeletons exhumed from a grave discovered in July 2007, and the results of a comprehensive genomic analysis of remains from the 1991 discovery. Additionally, ≈117 years old archival blood specimens from Nicholas II were obtained and genotyped, which provided critical material for the specific determination of individual identities and kinship identifications. Results of genotypic analyses of damaged historical specimens were evaluated alongside samples from descendants of both paternal and maternal lineages of the European Royal families, and the results conclusively demonstrate that the recently found remains belong to children of Nicholas II: Prince Alexei and his sister. The results of our studies provide unequivocal evidence that the remains of Nicholas II and his entire family, including all 5 children, have been identified. We demonstrate that convergent analysis of complete mitochondrial genome sequences combined with nuclear DNA profiles is an efficient and conclusive method for individual and kinship identification of specimens obtained from old historic relics.

One of the greatest mysteries for most of the twentieth century was the fate of the Romanov family, the last Russian monarchy. Following the abdication of Tsar Nicholas II, he and his wife, Alexandra, and their five children were eventually exiled to the city of Yekaterinburg. The family, along with four loyal members of their staff, was held captive by members of the Ural Soviet. According to historical reports, in the early morning hours of July 17, 1918 the entire family along with four loyal members of their staff was executed by a firing squad. After a failed attempt to dispose of the remains in an abandoned mine shaft, the bodies were transported to an open field only a few kilometers from the mine shaft. Nine members of the group were buried in one mass grave while two of the children were buried in a separate grave. With the official discovery of the larger mass grave in 1991, and subsequent DNA testing to confirm the identities of the Tsar, the Tsarina, and three of their daughters – doubt persisted that these remains were in fact those of the Romanov family. In the summer of 2007, a group of amateur archeologists discovered a collection of remains from the second grave approximately 70 meters from the larger grave. We report forensic DNA testing on the remains discovered in 2007 using mitochondrial DNA (mtDNA), autosomal STR, and Y- STR testing. Combined with additional DNA testing of material from the 1991 grave, we have virtually irrefutable evidence that the two individuals recovered from the 2007 grave are the two missing children of the Romanov family: the Tsarevich Alexei and one of his sisters.